Combination of sunitinib and 177Lu-labeled antibody cG250 targeted radioimmunotherapy: A promising new therapeutic strategy for patients with advanced renal cell cancer.

Sunitinib is an effective treatment for patients with metastatic Renal Cell Carcinoma (mRCC) but ultimately resistance occurs. The aim of this study was to investigate sunitinib resistance in RCCs and to develop therapeutic combination strategies with targeted radioimmunotherapy (RIT). We studied two RCC models, analyzed Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and AXL/MET expression and performed therapy studies in Balb/cnu/nu mice combining sunitinib and [177Lu]Lu-cG250 RIT (6.5 MBq/10 µg), specifically targeting RCC cells. pAXL and pMET were expressed in sunitinib-resistant SK-RC-52 and absent in sunitinib-sensitive NU12. NGS evaluation showed that expression of VEGFA, VEGFB, VEGFD, PGF and VEGFR1,2,3 was higher and expression of VEGFC and PDGFA was lower in NU12 than in SK-RC-52. Therapy studies combining sunitinib with [177Lu]Lu-cG250 RIT showed that the best response in mice with "resistant" SK-RC-52 tumors was observed with two cycles of Sunitinib and [177Lu]Lu-cG250 RIT, probably due to increased vascular permeability by sunitinib treatment. In the "sensitive" NU12 model, two cycles of [177Lu]Lu-cG250 RIT and two cycles of combination treatment were equally effective. Enhanced therapeutic efficacy was achieved when two agents ([177Lu]Lu-cG250 RIT and sunitinib) that on their own did not induce satisfactory response levels, are combined. Our findings provide a promising new therapeutic strategy for patients with advanced RCC.

Multimodal CEA-targeted fluorescence and radioguided cytoreductive surgery for peritoneal metastases of colorectal origin.

In patients with colorectal peritoneal metastases scheduled for cytoreductive surgery, accurate preoperative estimation of tumor burden and subsequent intraoperative detection of all tumor deposits remains challenging. In this study (ClinicalTrials.gov NCT03699332) we describe the results of a phase I clinical trial evaluating [111In]In-DOTA-labetuzumab-IRDye800CW, a dual-labeled anti-carcinoembryonic antigen (anti-CEA) antibody conjugate that enables both preoperative imaging and intraoperative radioguidance and fluorescence imaging. Primary study outcomes are safety and feasibility of this multimodal imaging approach. Secondary outcomes are determination of the optimal dose, correlation between tracer uptake and histopathology and effects on clinical strategy. Administration of [111In]In-DOTA-labetuzumab-IRDye800CW is well-tolerated and enables sensitive pre- and intraoperative imaging in patients who receive 10 or 50 mg of the tracer. Preoperative imaging revealed previously undetected lymph node metastases in one patient, and intraoperative fluorescence imaging revealed four previously undetected metastases in two patients. Alteration of clinical strategy based on multimodal imaging occurred in three patients. Thus, multimodal image-guided surgery after administration of this dual-labeled tracer is a promising approach that may aid in decision making before and during cytoreductive surgical procedures.

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Imaging carbonic anhydrase IX as a method for monitoring hypoxia-related radioresistance in preclinical head and neck cancer models.

BACKGROUND AND PURPOSE: Tumor hypoxia is an important cause of radioresistance and is associated with poor outcome.SPECT (Single-photon emission computed tomography) imaging enables visualizing tumor characteristics. We investigated the SPECT-radiotracer [111In]-girentuximab-F(ab')2 to image Carbonic Anhydrase IX (CAIX), an enzyme upregulated under hypoxic conditions. MATERIALS AND METHODS: Athymic mice with subcutaneous FaDu or SCCNij202 head and neck squamous cell carcinoma (HNSCC) xenografts were treated with atovaquone or were housed in a hypoxic chamber (8% O2). Next, [111In]-girentuximab-F(ab')2 was injected and 24 h later mice were euthanized for ex vivo biodistribution, autoradiography of the tumor, and immunohistochemical staining of the tumor. Tumor sections were analyzed for hypoxia, CAIX expression, vessels, and perfusion. Also, the effect of atovaquone on microSPECT scans was determined in the FaDu model. RESULTS: Atovaquone decreased CAIX expression by 69% (p = 0.017) compared with control tumors in FaDu, while in the SCCNij202 tumors no difference was observed. Hypoxic breathing did not increase CAIX expression or hypoxia staining in either tumor model, but did affect the necrotic tumor fraction. Ex vivo tracer uptake in the atovaquone treated group did not differ significantly from the control group, despite the difference in CAIX expression. Furthermore, SPECT imaging with [111In]-girentuximab-F(ab')2 did not discriminate atovaquone-treated versus control tumors. CONCLUSION: Atovaquone decreased CAIX expression only in the FaDu tumor model. [111In]-girentuximab-F(ab')2 specifically targets CAIX-expressing areas in HNSCC xenografts, but differences in vessel density and necrosis most likely affected tracer uptake in the tumors and therefore complicated quantification of changes in CAIX expression.

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Pyruvate-lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET.

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1-13 C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1-13 C]pyruvate to lactate conversion was followed by 13 C MRS. Subsequently [18 F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [18 F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant kpl of 13 C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [18 F]FDG PET measurements and kpl values for the xenografts of both tumor types. The kpl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [18 F]FDG-PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1-13 C]pyruvate.

Ex Vivo Assessment of Tumor-Targeting Fluorescent Tracers for Image-Guided Surgery.

Image-guided surgery can aid in achieving complete tumor resection. The development and assessment of tumor-targeted imaging probes for near-infrared fluorescence image-guided surgery relies mainly on preclinical models, but the translation to clinical use remains challenging. In the current study, we introduce and evaluate the application of a dual-labelled tumor-targeting antibody for ex vivo incubation of freshly resected human tumor specimens and assessed the tumor-to-adjacent tissue ratio of the detectable signals. Immediately after surgical resection, peritoneal tumors of colorectal origin were placed in cold medium. Subsequently, tumors were incubated with 111In-DOTA-hMN-14-IRDye800CW, an anti-carcinoembryonic antigen (CEA) antibody with a fluorescent and radioactive label. Tumors were then washed, fixed, and analyzed for the presence and location of tumor cells, CEA expression, fluorescence, and radioactivity. Twenty-six of 29 tumor samples obtained from 10 patients contained malignant cells. Overall, fluorescence intensity was higher in tumor areas compared to adjacent non-tumor tissue parts (p < 0.001). The average fluorescence tumor-to-background ratio was 11.8 ± 9.1:1. A similar ratio was found in the autoradiographic analyses. Incubation with a non-specific control antibody confirmed that tumor targeting of our tracer was CEA-specific. Our results demonstrate the feasibility of this tracer for multimodal image-guided surgery. Furthermore, this ex vivo incubation method may help to bridge the gap between preclinical research and clinical application of new agents for radioactive, near infrared fluorescence or multimodal imaging studies.

Targeting of radioactive platinum-bisphosphonate anticancer drugs to bone of high metabolic activity.

Platinum-based chemotherapeutics exhibit excellent antitumor properties. However, these drugs cause severe side effects including toxicity, drug resistance, and lack of tumor selectivity. Tumor-targeted drug delivery has demonstrated great potential to overcome these drawbacks. Herein, we aimed to design radioactive bisphosphonate-functionalized platinum (195mPt-BP) complexes to confirm preferential accumulation of these Pt-based drugs in metabolically active bone. In vitro NMR studies revealed that release of Pt from Pt BP complexes increased with decreasing pH. Upon systemic administration to mice, Pt-BP exhibited a 4.5-fold higher affinity to bone compared to platinum complexes lacking the bone-seeking bisphosphonate moiety. These Pt-BP complexes formed less Pt-DNA adducts compared to bisphosphonate-free platinum complexes, indicating that in vivo release of Pt from Pt-BP complexes proceeded relatively slow. Subsequently, radioactive 195mPt-BP complexes were synthesized using 195mPt(NO3)2(en) as precursor and injected intravenously into mice. Specific accumulation of 195mPt-BP was observed at skeletal sites with high metabolic activity using micro-SPECT/CT imaging. Furthermore, laser ablation-ICP-MS imaging of proximal tibia sections confirmed that 195mPt BP co-localized with calcium in the trabeculae of mice tibia.

[99mTc]-labelled interleukin-8 as a diagnostic tool compared to [18F]FDG and CT in an experimental porcine osteomyelitis model.

Osteomyelitis (OM) is an important cause of morbidity and sometimes mortality in children and adults. Long-term complications can be reduced when treatment is initiated in an early phase. The diagnostic gold standard is microbial examination of a biopsy and current non-invasive imaging methods are not always optimal. [111In]-leukocyte scintigraphy is recommended for peripheral OM, but is time-consuming and not recommended in children. [18F]FDG PET/CT is recommended for vertebral OM in adults, but has the disadvantage of false positive findings and a relatively high radiation exposure; the latter is a problem in children. [99mTc]-based tracers are consequently preferred in children. We, therefore, aimed to find a [99mTc]-marked tracer with high specificity and sensitivity for early detection of OM. Suppurating inflammatory lesions like OM caused by Staphylococcus aureus (S. aureus) will attract large numbers of neutrophils and macrophages. A preliminary study has shown that [99m Tc]-labelled IL8 may be a possible candidate for imaging of peripheral OM. We investigated [99mTc]IL8 scintigraphy in a juvenile pig model of peripheral OM and compared it with [18F]FDG PET/CT. The pigs were experimentally inoculated with S. aureus to induce OM and scanned one week later. We also examined leukocyte count, serum CRP and IL8, as well as performed histopathological and microbiological investigations. [ 99m Tc]IL8 was easily and relatively quickly prepared and was shown to be suitable for visualization of OM lesions in peripheral bones detecting 70% compared to a 100% sensitivity of [18F]FDG PET/CT. [ 99m Tc]IL8 is a promising candidate for detection of OM in peripheral bones in children.

A Clinical Feasibility Study to Image Angiogenesis in Patients with Arteriovenous Malformations Using 68Ga-RGD PET/CT.

Arteriovenous malformations (AVMs) have an inherent capacity to form new blood vessels, resulting in excessive lesion growth, and this process is further triggered by the release of angiogenic factors. 68Ga-labeled arginine-glycine-aspartate tripeptide sequence (RGD) PET/CT imaging may provide insight into the angiogenic status and treatment response of AVMs. This clinical feasibility study was performed to demonstrate that 68Ga-RGD PET/CT imaging can be used to quantitatively assess angiogenesis in peripheral AVMs. Methods: Ten patients with a peripheral AVM (mean age, 40 y; 4 men and 6 women) and scheduled for endovascular embolization treatment were prospectively included. All patients underwent 68Ga-RGD PET/CT imaging 60 min after injection (mean dose, 207 ± 5 MBq). Uptake in the AVM, blood pool, and muscle was quantified as SUVmax and SUVpeak, and a descriptive analysis of the PET/CT images was performed. Furthermore, immunohistochemical analysis was performed on surgical biopsy sections of peripheral AVMs to investigate the expression pattern of integrin αvß3Results:68Ga-RGD PET/CT imaging showed enhanced uptake in all AVM lesions (mean SUVmax, 3.0 ± 1.1; mean SUVpeak, 2.2 ± 0.9). Lesion-to-blood and lesion-to-muscle ratios were 3.5 ± 2.2 and 4.6 ± 2.8, respectively. Uptake in blood and muscle was significantly higher in AVMs than in background tissue (P = 0.0006 and P = 0.0014, respectively). Initial observations included uptake in multifocal AVM lesions and enhanced uptake in intraosseous components in those AVM cases affecting bone integrity. Immunohistochemical analysis revealed cytoplasmatic and membranous integrin αvß3 expression in the endothelial cells of AVMs. Conclusion: This feasibility study showed increased uptake in AVMs with angiogenic activity, compared with surrounding tissue without angiogenic activity, suggesting that 68Ga-RGD PET/CT imaging can be used as a tool to quantitatively determine angiogenesis in AVMs. Further studies will be conducted to explore the potential of 68Ga-RGD PET/CT imaging for guiding current treatment decisions and for assessing response to antiangiogenic treatment.

Follow-up imaging after cryoablation of clear cell renal cell carcinoma is feasible using single photon emission computed tomography with 111In-girentuximab.

PURPOSE: Detection of residual or recurrent vital renal tumor on follow-up (FU) cross-sectional imaging after ablative therapy is challenging. The specific and high expression levels of carbonic anhydrase IX (CAIX) in clear cell renal cell carcinoma (ccRCC) makes it a suitable target for imaging using radiolabeled anti-CAIX antibody girentuximab. The objective of this study was to evaluate the feasibility of targeted FU imaging 1 month after cryoablation of ccRCC using single photon emission computed tomography (SPECT) after 111In-labeled girentuximab administration. METHODS: In this prospective study 16 patients underwent 111In-girentuximab-SPECT before MR-guided renal cryoablation between February 2015 and September 2018. In case of tumor targeting 111In-girentuximab-SPECT was repeated 1 month following MR-guided cryoablation. Presence of residual or recurrent vital tumor was assessed on contrast-enhanced cross-sectional imaging during further FU. The standard FU imaging protocol consisted of MRI/CT scans at 1, 3, 6, 12, and 18 months and annually thereafter. RESULTS: A total of 10 (63%) patients showed positive tumor targeting on 111In-girentuximab-SPECT before cryoablation and 9 ( 56%) were eligible to undergo FU SPECT. Of the 9 111In-girentuximab-SPECT FU scans, 8 (89%) were considered negative. One (11%) scan showed uptake suggestive for residual vital tumor. Six months after treatment, FU CT showed contrast enhancement suggestive for residual/recurrent disease in the ablated zone at the site of the 111In-girentuximab uptake after treatment. During a mean FU of 21 months (range 1-33) no other cases with residual/recurrent disease were detected. CONCLUSION: FU imaging with 111In-girentuximab-SPECT is feasible after ccRCC cryoablation and may contribute to early detection of residual or recurrent disease.

Carcinoembryonic antigen-targeted photodynamic therapy in colorectal cancer models.

BACKGROUND: In colorectal cancer, survival of patients is drastically reduced when complete resection is hampered by involvement of critical structures. Targeted photodynamic therapy (tPDT) is a local and targeted therapy which could play a role in eradicating residual tumor cells after incomplete resection. Since carcinoembryonic antigen (CEA; CEACAM5) is abundantly overexpressed in colorectal cancer, it is a potential target for tPDT of colorectal cancer. METHODS: To address the potential of CEA-targeted PDT, we compared colorectal cancer cell lines with different CEA-expression levels (SW-48, SW-480, SW-620, SW-1222, WiDr, HT-29, DLD-1, LS174T, and LoVo) under identical experimental conditions. We evaluated the susceptibility to tPDT by varying radiant exposure and concentration of our antibody conjugate (DTPA-hMN-14-IRDye700DX). Finally, we assessed the efficacy of tPDT in vivo in 18 mice (BALB/cAnNRj-Foxn1nu/nu) with subcutaneously xenografted LoVo tumors. RESULTS: In vitro, the treatment effect of tPDT varied per cell line and was dependent on both radiant exposure and antibody concentration. Under standardized conditions (94.5 J/cm2 and 0.5 µg/µL antibody conjugate concentration), the effect of tPDT was higher in cells with higher CEA availability: SW-1222, LS174T, LoVo, and SW-48 (22.8%, 52.8%, 49.9%, and 51.9% reduction of viable cells, respectively) compared to cells with lower CEA availability. Compared to control groups (light or antibody conjugate only), tumor growth rate was reduced in mice with s.c. LoVo tumors receiving tPDT. CONCLUSION: Our findings suggest cells (and tumors) have different levels of susceptibility for tPDT even though they all express CEA. Furthermore, tPDT can effectively reduce tumor growth in vivo.

CAIX-targeting radiotracers for hypoxia imaging in head and neck cancer models.

Hypoxia-induced carbonic anhydrase IX (CAIX) expression is a prognostic marker in solid tumors. In recent years many radiotracers have been developed, but a fair comparison of these compounds is not possible because of the diversity in tumor models and other experimental parameters. In this study we performed a direct in vivo comparison of three promising CAIX targeting radiotracers in xenografted head and neck cancer models. The biodistribution of [111In]In-DOTA-ZCAIX:2 was directly compared with [111In]In-DTPA-G250-F(ab')2 and [111In] In-DTPA-G250 in female BALB/C nu/nu mice bearing two HNSCC xenografts with different levels of CAIX expression. In vivo biodistribution was quantified by means of microSPECT/CT scans and ex vivo biodistribution was determined with the use of a γ-counter. Tumors were snap frozen and sections were stained for CAIX expression, vessels, hypoxia (pimonidazole) and tumor blood perfusion. Tracer uptake was significantly higher in SSCNij153 tumors compared to SCCNij185 tumors for [111In]In-DOTA-HE3-ZCAIX:2: 0.32 ± 0.03 versus 0.18 ± 0.01%ID/g,(p = 0.003) 4 h p.i., for [111In]In-DTPA-girentuximab-F(ab')2: 3.0 ± 0.5%ID/g and 1.2 ± 0.1%ID/g (p = 0.03), 24 h p.i. and for [111In]In-DTPA-girentuximab: 30 ± 2.1%ID/g and 7.0 ± 1.0%ID/g (p = 0.0002) 72 h p.i. SPECT imaging with both [111In]In-DTPA-girentuximab-F(ab')2 and [111In]In-DTPA-girentuximab showed a clear difference in tracer distribution between the two tumor models. The whole IgG, i.e. [111In]In-DTPA-girentuximab, showed the highest tumor-to-muscle ratio. We showed that different CAIX-targeting radiotracers can discriminate a low CAIX-expressing tumor from a high CAIX-expressing head and neck cancer xenografts model. In these hypoxic head and neck xenograft models [111In]In-DTPA-girentuximab showed the most promising results.

PSMA-targeting agents for radio- and fluorescence-guided prostate cancer surgery.

Despite recent improvements in imaging and therapy, prostate cancer (PCa) still causes substantial morbidity and mortality. In surgical treatment, incomplete resection of PCa and understaging of possible undetected metastases may lead to disease recurrence and consequently poor patient outcome. To increase the chance of accurate staging and subsequently complete removal of all cancerous tissue, prostate specific membrane antigen (PSMA) targeting agents may provide the surgeon an aid for the intraoperative detection and resection of PCa lesions. Two modalities suitable for this purpose are radionuclide detection, which allows sensitive intraoperative localization of tumor lesions with a gamma probe, and fluorescence imaging, allowing tumor visualization and delineation. Next to fluorescence, use of photosensitizers may enable intraoperative targeted photodynamic therapy to eradicate remaining tumor lesions. Since radiodetection and optical imaging techniques each have their own strengths and weaknesses, a combination of both modalities could be of additional value. Here, we provide an overview of recent preclinical and clinical advances in PSMA-targeted radio- and fluorescence-guided surgery of PCa.

A pretargeted multimodal approach for image-guided resection in a xenograft model of colorectal cancer.

BACKGROUND: Image-guided surgery may improve surgical outcome for colorectal cancer patients. Here, we evaluated the feasibility of a pretargeting strategy for multimodal imaging in colorectal cancer using an anti-carcinoembryonic antigen (CEA) x anti-histamine-succinyl-glycine (HSG) bispecific antibody (TF2) in conjunction with the dual-labeled diHSG peptide (RDC018), using both a fluorophore for near-infrared fluorescence imaging and a chelator for radiolabeling. METHODS: Nude mice with subcutaneous (s.c) CEA-expressing LS174T human colonic tumors and CEA-negative control tumors were injected with TF2. After 16 h, different doses of 111In-labeled IMP-288 (non-fluorescent) or its fluorescent derivative RDC018 were administered to compare biodistributions. MicroSPECT/CT and near-infrared fluorescence imaging were performed 2 and 24 h after injection. Next, the biodistribution of the dual-labeled humanized anti-CEA IgG antibody [111In]In-DTPA-hMN-14-IRDye800CW (direct targeting) was compared with the biodistribution of 111In-RDC018 in mice with TF2-pretargeted tumors, using fluorescence imaging and gamma counting. Lastly, mice with intraperitoneal LS174T tumors underwent near-infrared fluorescence image-guided resection combined with pre- and post-resection microSPECT/CT imaging. RESULTS: 111In-RDC018 showed specific tumor targeting in pretargeted CEA-positive tumors (21.9 ± 4.5 and 10.0 ± 4.7% injected activity per gram (mean ± SD %IA/g), at 2 and 24 hours post-injection (p.i.), respectively) and a biodistribution similar to 111In-IMP288. Both fluorescence and microSPECT/CT images confirmed preferential tumor accumulation. At post mortem dissection, intraperitoneal tumors were successfully identified and removed using pretargeting with TF2 and 111In-RDC018. CONCLUSION: A pretargeted approach for multimodal image-guided resection of colorectal cancer in a preclinical xenograft model is feasible, enables preoperative SPECT/CT, and might facilitate intraoperative fluorescence imaging.

Measuring the Pancreatic ß Cell Mass in Vivo with Exendin SPECT during Hyperglycemia and Severe Insulitis.

OBJECTIVE: Targeting the glucagon-like peptide-1 receptor with radiolabeled exendin is a very promising method to noninvasively determine the ß cell mass in the pancreas, which is needed to unravel the pathophysiology of type 1 and type 2 diabetes. The present study aimed to explore the effects of both hyperglycemia and insulitis on the uptake of exendin in a spontaneous type 1 diabetes mouse model, nonobese diabetic (NOD) mice. METHODS: NOD mice (n = 75, 7-21 weeks old) were injected intravenously with [111In]In-DTPA-exendin-3, and single-photon emission computed tomography (SPECT) images were acquired 1 h pi. The pancreatic accumulation of [111In]In-DTPA-exendin-3 was quantified in vivo using SPECT and by ex vivo counting and correlated to the ß cell mass (BCM). The influence of insulitis and hyperglycemia on the exendin uptake was assessed. RESULTS: The pancreas could be visualized longitudinally using SPECT. A linear correlation was found between the BCM (%) and pancreatic uptake (%ID/g) as measured by ex vivo counting (Pearson r = 0.64, p < 0.001), which was not affected by either insulitis (Pearson r = 0.66, p = 0.83) or hyperglycemia (Pearson r = 0.57, p = 0.51). Biodistribution and ex vivo autoradiography revealed remaining [111In]In-DTPA-exendin-3 uptake in the pancreas despite total ablation of BCM. CONCLUSIONS: Despite hyperglycemia and severe insulitis, we have found a good correlation between BCM and pancreatic exendin uptake, even in a suboptimal model with relatively high background activity.

Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG-PET/CT in patients with newly diagnosed metastatic renal cell carcinoma.

PURPOSE: The main objective of this preliminary analysis of the IMaging PAtients for Cancer drug selecTion (IMPACT)-renal cell cancer (RCC) study is to evaluate the lesion detection of baseline contrast-enhanced CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT in detecting ccRCC lesions in patients with a good or intermediate prognosis metastatic clear cell renal cell carcinoma (mccRCC) according to the International Metastatic Database Consortium (IMDC) risk model. METHODS: Between February 2015 and March 2018, 42 newly diagnosed mccRCC patients with good or intermediate prognosis, eligible for watchful waiting, were included. Patients underwent CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT at baseline. Scans were independently reviewed and lesions of ≥10 mm and lymph nodes of ≥15 mm at CT were analyzed. For lesions with [89Zr]Zr-DFO-girentuximab or [18F]FDG-uptake visually exceeding background uptake, maximum standardized uptake values (SUVmax) were measured. RESULTS: A total of 449 lesions were detected by ≥1 modality (median per patient: 7; ICR 4.25-12.75) of which 42% were in lung, 22% in lymph nodes and 10% in bone. Combined [89Zr]Zr-DFO-girentuximab-PET/CT and CT detected more lesions than CT alone: 91% (95%CI: 87-94) versus 56% (95%CI: 50-62, p = 0.001), respectively, and more than CT and [18F]FDG-PET/CT combined (84% (95%CI:79-88, p < 0.005). Both PET/CTs detected more bone and soft tissue lesions compared to CT alone. CONCLUSIONS: The addition of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT to CT increases lesion detection compared to CT alone in newly diagnosed good and intermediate prognosis mccRCC patients eligible for watchful waiting.

Direct comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for 89Zr-immunoPET.

PURPOSE: Currently, the most commonly used chelator for labelling antibodies with 89Zr for immunoPET is desferrioxamine B (DFO). However, preclinical studies have shown that the limited in vivo stability of the 89Zr-DFO complex results in release of 89Zr, which accumulates in mineral bone. Here we report a novel chelator DFOcyclo*, a preorganized extended DFO derivative that enables octacoordination of the 89Zr radiometal. The aim was to compare the in vitro and in vivo stability of [89Zr]Zr-DFOcyclo*, [89Zr]Zr-DFO* and [89Zr]Zr-DFO. METHODS: The stability of 89Zr-labelled chelators alone and after conjugation to trastuzumab was evaluated in human plasma and PBS, and in the presence of excess EDTA or DFO. The immunoreactive fraction, IC50, and internalization rate of the conjugates were evaluated using HER2-expressing SKOV-3 cells. The in vivo distribution was investigated in mice with subcutaneous HER2+ SKOV-3 or HER2- MDA-MB-231 xenografts by PET/CT imaging and quantitative ex vivo tissue analyses 7 days after injection. RESULTS: 89Zr-labelled DFO, DFO* and DFOcyclo* were stable in human plasma for up to 7 days. In competition with EDTA, DFO* and DFOcyclo* showed higher stability than DFO. In competition with excess DFO, DFOcyclo*-trastuzumab was significantly more stable than the corresponding DFO and DFO* conjugates (p < 0.001). Cell binding and internalization were similar for the three conjugates. In in vivo studies, HER2+ SKOV-3 tumour-bearing mice showed significantly lower bone uptake (p < 0.001) 168 h after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (femur 1.5 ± 0.3%ID/g, knee 2.1 ± 0.4%ID/g) or [89Zr]Zr-DFO*-trastuzumab (femur 2.0 ± 0.3%ID/g, knee 2.68 ± 0.4%ID/g) than after injection with [89Zr]Zr-DFO-trastuzumab (femur 4.5 ± 0.6%ID/g, knee 7.8 ± 0.6%ID/g). Blood levels, tumour uptake and uptake in other organs were not significantly different at 168 h after injection. HER2- MDA-MB-231 tumour-bearing mice showed significantly lower tumour uptake (p < 0.001) after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (16.2 ± 10.1%ID/g) and [89Zr]Zr-DFO-trastuzumab (19.6 ± 3.2%ID/g) than HER2+ SKOV-3 tumour-bearing mice (72.1 ± 14.6%ID/g and 93.1 ± 20.9%ID/g, respectively), while bone uptake was similar. CONCLUSION: 89Zr-labelled DFOcyclo* and DFOcyclo*-trastuzumab showed higher in vitro and in vivo stability than the current commonly used 89Zr-DFO-trastuzumab. DFOcyclo* is a promising candidate to become the new clinically used standard chelator for 89Zr immunoPET.

Development and characterization of a theranostic multimodal anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors.

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light. Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA+) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm2) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions. Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm2 resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm2 (p = 0.0067) and 3x 50 J/cm2 (p = 0.0338). Principal conclusions:111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.